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RESEARC H ARTIC L E Open Access
Effects of short-term glucocorticoid treatment on
changes in cartilage matrix degradation and
chondrocyte gene expression induced by
mechanical injury and inflammatory cytokines
Yihong CS Lu
1
, Christopher H Evans
2
and Alan J Grodzinsky
1,3*
Abstract
Introduction: Traumatic joint injury damages cartilage and causes adjacent joint tissues to release inflammatory
cytokines, increasing the risk of developing osteoarthritis. The main objective of this study was to determine
whether the combined catabolic effects of mechanical injury, tumor necrosis factor alpha (TNFa) and interleukin-6
(IL-6)/soluble IL-6 receptor (sIL-6R) on cartilage could be abolished by short-term treatment with glucocorticoids
such as dexamethasone.
Methods: In an initial dexamethasone-dose-response study, bovine cartilage explants were treated with TNFa and
increasing concentrations of dexamethasone. Bovine and human cartilage explants were then subjected to
individual and combined treatments with TNFa, IL-6/sIL-6R and injury in the presence or absence of
dexamethasone. Treatment effects were assessed by measuring glycosaminoglycans (GAG) release to the medium
and synthesis of proteoglycans. Additional experiments tested whether pre-exposure of cartilage to
dexamethasone could prevent GAG loss and inhibition of biosynthesis induced by cytokines, and whether post-
treatment with dexamethasone could diminish the effects of pre-established cytokine insult. Messenger ribonucleic
acid (mRNA) levels for genes involved in cartilage homeostasis (proteases, matrix molecules, cytokines, growth and
transcription factors) were measured in explants subjected to combined treatments with injury, TNFa and
dexamethasone. To investigate mechanisms associated with dexam ethasone regulation of chondrocyte metabolic
response, glucocorticoid receptor (GR) antagonist (RU486) and proprotein convertase inhibitor (RVKR-CMK) were
used.
Results: Dexamethasone dose-dependently inhibited GAG loss and the reduction in biosynthesis caused by TNFa.
The combination of mechanical injury, TNFa and IL-6/sIL-6R caused the most severe GAG loss; dexamethasone

reduced this GAG loss to control levels in bovine and human cartilage. Additionally, dexamethasone pre-treatment
or post-treatment of bovine explants lowered GAG loss and increased proteoglycan synthesis in cartilage explants
exposed to TNFa. Dexamethasone did not down-regulate aggrecanase mRNA levels. Post-transcriptional regulation
by dexamethasone of other genes associated with responses to injury and cytokines was noted. GR antagonist
reversed the effect of dexamethasone on sulfate incorporation. RVKR-CMK significantly reduced GAG loss caused by
TNFa + IL-6 + injury.
Conclusions: Short-term glucocorticoid treatment effectively abolished the catabolic effects exerted by the
combination of pro-inflammatory cytokines and mechanical injury: dexamethasone prevented proteoglycan
degradation and restored biosynthesis. Dexamethasone appears to regulate the catabolic response of chondrocytes
* Correspondence:
1
Department of Biological Engineering, MIT, 500 Technology Square NE47-
377, Cambridge, MA, 02139, USA
Full list of author information is available at the end of the article
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>© 2011 Lu et al.; licens ee BioMed Central Ltd. Thi s is an open access article distributed under the terms of the Creative Commons
Attribution License ( which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
post-transcriptionally, since the abundance of transcripts encoding aggrecanases was still elevated in the presence
of dexamethasone.
Introduction
Osteoarthritis (OA) is characterized by chronic, irrever-
sible degradation of articular cartilage. Traumatic joint
injury in young adults greatly increases the risk of devel-
oping OA [1,2] and post-traumatic OA remains a major
clinical and societal problem. Treatments following joint
trauma initially focus on reducing pain and swelling,
and often by subsequent reconstru ctive surgery to stabi-
lize joint b iomechanics, for example, for injuries invol-
ving anterior cruciate ligament (ACL) rupture. However,

these interventions do not prevent the progression to
secondary OA after injury [3,4]. Following knee injury,
high levels of aggrecan fragments and cross-linked pep-
tides from type II collagen accumulate in the synovial
fluid [5]. Moreover, joint injury results in an immediate
surge in synovial f luid concentrations of pr o-inflamma-
tory cytokines, including tumor necrosis factor-a
(TNFa), interleukin-1b (IL-1b), IL-6 and IL-8 [6-8]. The
levels of these cytokines remain elevated for weeks and
eventually decrease to levels detected in chronic OA
joints [8]. Thus, cartilage in the injured joint is often
subjected to an initial biomechanical insult [9] and then
further compromised by the presence of high levels of
inflammatory cytokines [10].
In a recent report, we highlighted the interplay
between mechanical and cytokine-mediated pathways
regulating cartilage degradation relevant to traumatic
joint injury [11]. We used an in vitro model involving
injurious compression of cartilage explants to simulate
the initial mechanical insult, and subsequent co-culture
with exogenous cytokines to simulate the inflammatory
component. In both human and bovine cartilage,
mechanical injury and TNFa synergist ically increased
proteoglycan degradation [11]. Moreover, mechanical
injury potentiated the combined catabolic effects of
TNFa and IL- 6 along with its soluble receptor, sIL-6R,
causing the most severe glycosaminoglycan (GAG) loss
among all treatment conditions. Proteoglycan degrada-
tion was found to be mediated by aggrecanase activity
[11] in these studies.

In the present study, we address the potential utility of
glucocorticoids (GCs) in the treatment of joint injury.
Intra-articular injection of GCs is an established treat-
ment for both chronic OA and rheumatoid arthritis
(RA) [12,13]. GCs exert their effects by binding to intra-
cellular glucocorticoid receptors (GRs), which act as
transcription factors in cells. The activated GRs either
directly or indirectly regulate the transcription of target
genes. For example, GRs are known to enhance the
production of anti-inflammatory cytokines such as IL-1
receptor antagonist and IL-10 [14], while the expression
of molecules associated with inflammatory processes,
including cytokines IL-1b,IL-6,TNFa, and cyclooxy-
genase-2 [15-18] is repressed. The effects of GCs in car-
tilage are less well understood. Since human
chondrocytes have been shown to express GRs [19,20],
the potential effects of GCs in treating joint disorders
may be due to direct regulation of chondrocytes, but
this possibility has not been widely studied.
Dexamethasone (DEX) is a very potent s ynthetic GC
due to its high receptor binding affinity [21]. DEX has
been commonly used in cartilage tissue engineering;
numerous studies have demonstrated that DEX potenti-
ates the ability of progenitor cells to undergo chondro-
genic differentiation and to synthesize cartilage
proteoglycans [22-24]. However, the effects of DEX on
cartilage matrix turnover, particularly those changes
associated with joint injury, remain unclear.
The objectives of this study were: (1) to test the hypoth-
esis that short-term treatment with DEX could abolish

matrix degradation and the known reduction of chondro-
cyte biosynthesis caused by the combination of mechanical
injury and inflammatory cytokines in bovine and human
cartilage explants, (2) to investigate whether DEX regulates
this metabolic response at the transcriptional level in
chondrocytes, and (3) to explore mechanistic pathways by
which DEX may suppress cartilage degradation. The path-
ways of interest included regulation of aggrecanase gene
expression and the activation of aggrecanases by propro-
tein convertases, the effects of DEX on inducible nitric
oxide synthase (iNOS) mRNA and protein levels, and the
role of glucocorticoid receptors.
A disintegrin and metalloproteinase with thrombos-
pondin motifs-4,-5 (ADAMTS-4 and -5) are the primary
aggrecanases responsible for the pathological process of
aggrecan degradation in human OA [25]. Aggrecanases
are synthesized as laten t, inactive enzymes whose pro-
domains must be removed by proprotein convertases
(PCs) in order to express their catalytic function. Studies
have shown increased activity of PCs in both osteoar-
thritic and cytokine-stimulated cartilage, and inhibiting
PC activity significantly reduced cytokine-induced aggre-
can degradation [26]. Among the PCs, furin, PACE4 and
PC5/6 are capab le of removing the prodomain o f
ADAMTS-4 [27], while furin and PC7 have been shown
to process pro-ADAMTS-5 [28]. Thus, regulation of
aggrecanase activation as well as mRNA levels of
ADAMTS-4 and -5 are both pathways of interest.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 2 of 15

Materials and methods
After a description of cartilage explant harvest and the
methods for applying injurious mechanical compr ession
to these explants, we t hen delineated methods to test
the effects DEX on matrix metabolism in explants sub-
jected to mechanical injury and i nflammatory cytokine
challenge. In one series of experiments using bovine and
human cartilage, DEX was added immediately at the
time of injury and cytokine treatment. In anoth er series
of experiments using bovine tissue, DEX was added
either two d ays before or two days afte r injury + cyto-
kine treatment to test whether DEX could protect and/
or rescue changes in cartilage matrix metabolism caused
by injury. The concentration of DEX used in all these
tests was determined from an initial dose-response
study. We then describe methods for experiments focus-
ing on mechanistic pathways, including studies of DEX
regulation of chondrocyte transcription, effects of DEX
on iNOS mRNA and protein levels, and inhibition of
glucocorticoid receptors and proprotein convertases.
Bovine cartilage harvest and culture
Cartilage disks were harvested from the femoropatellar
grooves of one- to two-week-old bovine calf knee joints
(obtained from Research 87, Hopkinton, MA, USA) as
previously described [29]. A total of 16 joints from 13
different animals and 1 human were used. Briefly, carti-
lage-bone cylinders (9 mm diamet er) were cored per-
pendicular to the surface. After a level surface was
obtained by removing the most superficial layer
(approximately 100 to 200 μm), one to two sequential 1

mm-slices of middle zone cartilage were cut from each
cylinder. Five di sks (3 mm-diameter, 1 mm-thick) were
cored from each slice using a dermal punch. Cartilage
from this middle zone in newborn calves was shown
previously to have a reasonably homogeneous popula-
tion of cells and matrix [30]. Cartilage disks for all treat-
ment groups were matched for depth and location along
the joint surface [31]. Disks were equilibrated in serum-
free medium (low-glucose DMEM (Cellgro, Herndon,
VA, USA)), 10 mM HEPES buffer (Invitrogen, Carlsbad,
CA, USA), supplemented with 1% insulin-transferrin-
selenium (10 μg/ml, 5.5 μg/ml and 5 ng/ml, respec-
tively), 0.1 mM nonessential amino acids, 0.4 mM pro-
line, 20 μg/mL ascorbic acid, 100 units/mL penicillin G,
100 μg/mL streptomycin, and 0.25 μg/mL amphotericin
B (all from Sigma, St. Louis, MO, USA)) for two days
prior to treatment in a 37°C, 5% CO
2
incubator.
Postmortem adult human donor tissue
Human donor knee cartilage (49-yr-old female, modi-
fied-Collins [32] grade-1 knee joint) was obtain ed from
theGiftofHopeOrganandTissueDonorNetwork
(Elmhurst, IL, USA), approved by the Office of Research
Affairs at Rush-Presbyterian-St. Luke’s Medical Center
and the Committee on Use of Humans as Experimental
Subjects at MIT. Any fibrillated areas detected under
visual inspection were excluded from the study. Human
cartilage harvest and culture were identical to that of
bovine, but included the intact superficial zone and each

disk was approximately 0.8 mm thick. Human knee car-
tilage was obtained from both the femoropatellar groove
and femoral condyles.
Injurious compression
After equilibration in medium for three days, disks were
injuriously compressed in a custom-designe d incubator-
housed apparatus [33,34]. Each bovine disk was sub-
jected to radially unconfined compression to 50% final
strain at 1 mm/second velocity (100% per second strain
rate), followed by immediate release of load at the same
rate, as described [29]. Immediately after injury, some
disks were deformed to an ellipsoidal shape (deforma-
tion score of 1 or 2 as described in [35]), but none
exhibited gross fissuring. Adult human cartilage disks
were thinner, had intact superficial zone and different
effective biomechanical behavior compared to the imma-
ture bovine disks, reflecting the anisotropy and inhomo-
geneity associated with the presence of the superficial
zone. The combined properties were such that higher
strain and strain rate values were needed to produce
levels of peak stress and visible deformation in human
cartilage similar to that observed for immature bovine
tissue [29]. Thus, a strain of 60% and strain rate of
300%/second were used , the same values utilized in our
recent report with this in vitro injury plus cytokine sti-
mulation system for adult human cartilage [11]. The
resulting macroscopic tissue changes in human cartilage
disks were similar (elliptical appearance) to those
described previously using our human cartilage injury
model and scoring system [36]. After injury, samples

were immediately placed in treatment medium.
DEX dose-response
In a DEX dose-response study, bovine cartila ge samples
(70 disks from two joints of one animal) were treated
either with or without rhTNFa (25 ng/mL) and incu-
bated for six days with increasing concentrations of
DEX (Sigma, St. Louis, MO, USA), from 0.1 nM to 100
μM.
Exogenous cytokines, injury and DEX treatments
Cartilage samples were either subjected to injurious
compression or left uninjured, incubated with or with-
out cytokines (all from R&D Systems, Minneapolis, MN,
USA), and with or without DEX. Previously [11], we
observed that treatments with TNFa,TNFa + injury,
TNFa + IL-6/sIL-6R, and TNFa + injury + IL-6/sIL-6R
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 3 of 15
caused significant release of GAGs from both human
and bovine cartilage explants, with the latter condition
causing the most severe loss of GAG. In this study, we
first examined the effects o f DEX on cartilage explants
under these same conditions. For bovine cartilage (70
disks from two joints of another animal), DEX and
recombinant human TNFa (rhTNFa)wereusedat10
nM and 25 ng/mL, respectively, based on the results
from the DEX dose response study. For human cartilage
(36 disks from the distal femur), DEX and rhTNFa were
used at 100 nM and 100 ng/mL, respectively. rhIL-6 (50
ng/mL)wasalwaysusedincombinationwithsoluble
IL-6 receptor (sIL-6R, 250 ng/mL), since this combina-

tion was found previously to induce greater aggrecan
degradation than when used separately in the presence
of TNFa [37]. Bovine cartilage disks were cultured in
these conditions for six days. Culture duration for
human explants was extended to 10 days based on ear-
lier studies showing that human cartilage released sGAG
more slowly than bovine cartilage for these conditions
[11]. Medium was replaced every two days and saved for
analysis.
Pre- and post-treatment with DEX
To test whether a short-duration pre-exposure of carti-
lage to DEX could prevent GAG loss and inhibition of
biosynthesis induced by subsequent cytokine treatment,
bovine cartilage disks (10 disks from a separate animal)
were either pre-treated with DEX for two days or incu-
bated in medium alone. Afterwards, both groups were
incubated in medium containing TNFa but no DEX for
an additional four days. To test whether post-treatment
with DEX could diminish the effects of a pre-established
cytokine insult, cartilage explants (10 disks from a dif-
ferent animal) were first treated with TNFa for two
days, and DEX was then added to the medium in addi-
tion to continued treatment with TNFa for another four
days. GAG loss and radiolabel incorporation were mea-
sured as above.
Matrix biosynthesis and biochemical analyses
Two days before termination of the bovine cultures, the
medium of each disk was supplemented with 5 μCi/ml
Na
2

35
SO
4
(Perkin-Elmer, Norwalk, CT, USA) as a mea-
sure of the rate of proteoglycan synthesis. The amount
of radiolabeled sulfate was doubled in studies of human
cartilage. Upon termination, disks were washed, weighed
and solubilized (proteinase K, Roche, Indianapolis, IN,
USA), and radiolabel incorp oration was measured using
a liquid scintillatio n counter [30]. The amounts of GAG
lost to the medium and retained in the cartilage were
measured using the dimethylmethylene blue (DMMB)
assay, with shark chondroitin sulfate (Sigma) as the
standard [38].
Gene expression studies: RNA extraction and real-time
PCR
To examine the effects of DEX, injury and TNFa on
chondrocyte gene expression, bovine cartilage disks
from six different animals were cultured for four day s
under the eight treatment conditions: (1) no-treatment
control, (2) DEX-only, (3) mechanical injury only, (4)
DEX + injury, (5) TNFa, (6) TNFa + DEX, (7) TNFa +
injury, and (8) TNFa + injury + DEX. A total of 48
disks per animal from each of six different joints (six
different animals) were used. From each joint, RNA was
pooled from the six disks assigned to each of the e ight
treatment conditions (matching disks from along the
joint surface across treatment groups). Thus, there were
six different repeats of this experiment in total, with
each repeat corresponding to a different joint (animal).

Samples were pulverized in liquid nitrogen and homoge-
nized in TRIzol reagent (Invitrogen). The extract was
spun at 13,000 g for 10 minutes in Phase Gel tubes
(Eppendorf, Hamburg, Germany) with 10% chloroform
(Sigma). After spinning, the clear supernatant was
obtained and RNA was isolated using the RNeasy Mini
columns (Qiagen, Chatsworth, CA, USA); genomic DNA
was removed by a DNase digestion step (Qiagen)
according to the manufacturer’ s protocol. Absorbance
was read at 260 nm and 280 nm to measure the concen-
tration of RNA and the purity of the extract. Reverse
transcription of equal quantities of RNA (2.5 μg) from
each condition was performed using the AmpliTaq-Gold
Reverse Transcription Kit (Applied Biosystems, Foster
City, CA, USA) [39]. Genes of interests were those
involved in cartilage homeostasis, including matrix
molecules (aggrecan, collagen II and IX), cytokines (IL-
1b,IL-6,TNFa), proteases and protease inhibitors
(ADAMTS-4,-5, matrix metalloproteinase-3 (MMP-3),
tis sue inhibitor of metalloproteinase-3 (TIMP-3)), iNOS
and a housekeeping gene (18 S). Bovine primer
sequences for all genes except iNOS, collagen IX and
IL-6 were reported in our previous studies [40,41];
sequences for these latter three genes were reported in
another study [42]; they were also designed using Pri-
mer3 software [43] on the basis of bovine sequences. A
standard curve for amplification was generated for each
of the primer. All primers demonstrated approximately
equally efficiency, with standard curve slopes of approxi-
mately 1, in dicating a doubling in compl ementary DNA

quantity in each cycle [39]. Real-time PCR was per-
formed using Applied Biosystems ABI 7900HT instru-
ment and SYBR Green Master Mix (Applied
Biosystems). Measured threshold values (Ct) were con-
verted to RNA copy number according t o primer effi-
ciencies. Within each condition, the RNA copy numbers
for each gene were normalized to that of 18 S from the
same condition. To examine the effects of treatments,
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 4 of 15
each gene was then normalized to its level in the no-
treatment control group.
Pathways: inhibition of glucocorticoid receptor,
proprotein convertases and iNOS
TheroleofchondrocyteGRsintheresponsetoDEX
was studied in bovine cartilage samples (30 disks from
one animal) by treatment with the GR antagonist,
RU486 (1 μM, Sigma), in the presence of TNFa and
TNFa + DEX for six days. The role of proprotein con-
vertases in matrix degradation was tested by the addi-
tion of the PC inhibitor decanoyl-RVKR-CMK (10 μM,
Calbiochem, La Jolla, CA, USA) to bov ine cartilage
explants (35 disks from one animal) cultured with differ-
ent combinations of TNFa, IL-6/sIL-6R and mechanica l
injury. The levels of iNOS protein were measured fol-
lowing four-day treatments of bovine cartilage disks
with TNFa ± injury, in the presence or absence of DEX.
The disks were then pulverized in liquid nitrogen and
homogenized in buffer solution (20 mM pH 7.6 Tris,
120 Mm NaCl, 10 mM EDTA, 10% glycerol, 1% Nonidet

P-40 (Sigma) with protease inhibitor cocktail (Roche)).
Equal amounts of protein were collected from each con-
dition, run on 4 to 15% gels (Invitrogen) and then trans-
ferred to polyvinylidene difluoride (BioRad, Hercules,
CA, USA) for immunoblotting. Western blots were per-
formed using anti-bovine iNOS antibody (1:1000, Milli-
pore, Billerica, MA, USA), followed by secondary
antibodi es conjugated to horseradish peroxidase (1:4000,
Cell Signaling Technology, Beverly, MA, USA). In
another study, nitrite levels in the medium of 48 disks
(one animal) were analyzed using the Griess Reagents
(Invitrogen).
Statistical analyses
In studying the effect of DEX dose on GAG loss and
proteoglycan biosynthesis, a general linear model was
used to analyze the data, followed by Dunnet’stestfor
comparisons to controls. In evaluating the effect of DEX
on GAG loss, sulfate incorporation and nitrite accumu-
lation in cytokine -treated and mechanically-i njured
bovine and human cartilage, as well as the effect of
CMK on GAG loss in bovine cartilage, a general linear
model with Bonferroni’ s test was used to conduct
hypothesis-based comparisons. For the study testing the
effect of RU486, a general linear model was used fol-
lowed with Tukey’s test. In the studies of pre- and post-
treatment of cartilage with DEX, a two-way general lin-
ear model with Tukey’s test was used to evaluate differ-
ences between conditions and time points. For gene
expression studies, log-transformed expression data
were analyzed using a general linear model followed by

Dunnet’s test for comparison of each of the conditions
to no-treatment controls. All values are expressed as
mean ± SEM, with P ≤0.05 taken as statistically signifi-
cant. Statistical analyses were performed using SYSTAT-
12 software (Richmond, CA, USA).
Results
DEX dose-dependently inhibited GAG loss and reversed
the reduction in chondrocyte biosynthesis induced by
TNFa-treatment of bovine cartilage
Experiments were performed to test the effect of DEX
(0.1 nM to 100 μM) on both TNFa-treated and
untreated control cartilage explants (Figure 1). TNFa
treatment significantly increased GAG loss to the med-
ium (to 16.2 ± 0.5% of total by six days) compared to
that from controls (8.5 ± 0.2%, mean ± SEM), a finding
consistent with previous studies [11]. DEX at concentra-
tions of 1 nM or higher reduced GAG loss induced by
TNFa treatment to levels that were not significantly dif-
ferent from controls. At concentration s 100 nM and
higher, DEX treatment alone suppressed GAG loss to
levels below those found in control cultures (Figure 1A).
All cartilage samples from Figure 1A were also radi-
olabele d with
35
S-sulfate to measure the rates of proteo-
glycan biosynthesis in response to treatment conditions.
Compared to controls (having
35
S-sulfate incorporation
= 51.6 ± 2.1 pmol/hour/mg wet weight), TNFa treat-

ment significantly reduced sulfate incorporation to 25.3
± 2.3 pmol/hour/mg (Figure 1B). In contrast, treatment
with TNFa and DEX at concentrations of 0.1 nM and
higher showed sulfate incorporation rates, which were
not significantly different from controls. Moreov er, con-
centrations of 0.1 μM to 100 μMDEXalonesignifi-
cantly increased sulfate incorporation above control
levels (70.0 ± 1.6, 75.9 ± 3.5, and 73.0 ± 1.0 pmol/h/mg,
respectively, Figure 1B).
DEX inhibited GAG loss and biosynthesis reduction in
bovine cartilage treated with combinations of mechanical
injury, TNFa and IL-6/sIL6R
Consistent with our previous findings, TNFa treatment
together with mechanical injury or IL-6/sIL-6R or the
combination of all three treatments, significantly
increased GAG release from bovine cartilage (Figure
2A) [11]. The combined treatment with injury +TNFa
IL-6/sIL-6R caused the most severe GAG loss by six
days. The addition of 10 nM DEX significantly reduced
GAG loss caused by injury +TNFa,TNFa +IL-6/sIL-
6R, and injury +TNFa + IL-6/sIL-6R, the latter from
53.6 ± 9.8% down to 13.8 ± 1.5% compared to 7.3 ±
0.2% for controls.
TNFa treatment, either alone or together with
mechanical injury, IL-6/sIL-6R or their combination,
greatly reduced sulfate incorporation rates (Figure 2B),
as seen in our previous study [11]. Importantly, DEX
abolished the reduction in biosynthesis caused by all
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 5 of 15

these treatments. For example, treatment with TNF a +
IL-6/sIL-6R + injury reduced sulfate incorporation to
26.2 ± 7.2 p mol/hour/mg, whereas the addition of 10
nM DEX to this same condition significantly increased
sulfate incorporation to 96.2 ± 13.44 pmol/hour/mg, a
level that was not significantly different from no-treat-
ment controls.
DEX treatment reduced GAG loss in human cartilage
explants
Treatment with injury + TNFa +IL-6/sIL-6Rgreatly
increased GAG loss from human knee cartilage (to 36.0
± 4% of total, Figure 3), consistent with our previous
report [11]. Under these conditions, the addition of 100
nM DEX significantly reduced GAG loss to 20.5 ± 1.5%,
but showed no effect on sulfate incorp oration (data not
shown).
Pre-treatment with DEX reduced GAG loss and increased
sulfate incorporation in TNFa-treated cartilage
Bovine cartilage samples were pre-incubated with 10 nM
DEX for two days and then cultured in medium con-
taining TNFa, wit hout DEX, for an additional four days.
The pre-treatment with DEX significantly reduced
TNFa-induced GAG loss by Day 4 (Figure 4A), and sig-
nificantly increased the sulfate incorporation rate com-
pared to the condition without DEX pre-treatment
(Figure 4B).
0
5
10
15

20
TNFα (25ng/mL)
DEX (nM)
-
-
-
0.1
1
10
100
10
3
- -
-
0.1
1
10
100
-
++
+
++++
GAG Loss from Bovine Cartilag
e
(% of Total)
*
*
*
*
0

20
40
60
80
100
Sulfate Incorporation
(pmol/hr.mg)
T
NF
α
(25ng/mL)
-

-

*
*
++
+
++++
*
A.
B.
10
5
10
3
10
5
DEX (nM)

-
-
0.1
1
10
100
10
3
0.1
1
10
100
10
5
10
3
10
5
*
*
Figure 1 Dexamethasone dose-respose studies. A) Effec t of DEX on TNFa-stimulated GAG loss in bovine cartilage explants. Cartilage tissues
were cultured in DEX (0.1 nM-100 μM)-supplemented media, with or without TNFa (25 ng/ml) for six days. The total GAG content of untreated
control cartilage was 465.6 ± 23.1 μg GAG/disk (mean ± SEM). DEX, at 1 nM and higher reduced GAG loss induced by TNFa treatment. B) Effect
of DEX on chondrocyte biosynthetic rates as measured by
35
S-sulfate incorporation during days 4 to 6. TNFa treatment significantly lowered
biosynthesis of sulfated proteoglycans; DEX reversed this inhibition at concentrations of 0.1 nM or higher. Values in A and B are presented as
mean ± SEM; n = 5 cartilage disks per condition. *= P < 0.05 vs. no-treatment control. DEX, dexamethasone; GAG, glycosaminoglycans; SEM,
standard error of the mean; TNFa, tumor necrosis factor alpha.
Lu et al. Arthritis Research & Therapy 2011, 13:R142

/>Page 6 of 15
Post-treatment with DEX reduced GAG loss and increased
sulfate incorporation in TNFa-treated cartilage
We next examined whether DEX would exert anti-cata-
bolic effects in cartilage samples where matrix degrada-
tion had already been induced by cytokine stimulation.
All cartilage samples were pre-incubated with TNFa for
two days (st arting at Day -2 in Figure 4C). Afterwards,
starting at Day 0, one group of samples was cultured in
medium with TNFa +10 nM DEX, while a second
group was treated w ith TNFa alone. After the two-day
pre-incubation with TNFa, disks from both groups had
lost approximate ly 6% of total GAG (Day 0, Figure 4C).
The addition of D EX significantly a ttenuated GAG loss
and increased proteoglycan biosynthesis by Day 4 (Fig-
ure 4C, D).
The anti-catabolic effects of DEX were glucocorticoid
receptor (GR) mediated
To assess whether the inhibition of GAG loss and the
increase in proteoglycan biosynthesis in DEX-treated
cartilage were GR mediated, bovine explants were trea-
ted with the GR antago nist RU486 in addition to TNFa
±DEX.AsshowninFigure5,TNFa significantly
increased GAG loss and reduced sulfate incorporation
DEX (10nM)

- +++++
GAG Loss from Bovine Cartilage
(% of Total)
0

20
40
60
80
*
*
*
0
40
80
120
160
Sulfate Incorporation
(pmol/hr.mg)
*
*
*
*
A.
B.
TNFα (25ng/mL)
Mechanical Injury
IL-6/sIL-6R (50/250 ng/mL)
-
-
+
+
+
+
+

+
+
+
+
+
+
+
-
-
-
-
-
-
+
+
+
+
-
-
-
-
-
-
-
++ + + +
-
-
+
+
+

+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
+
+
+
+
-
-
-
-
-
-
DEX (10nM)

TNFα (25ng/mL)
Mechanical Injury
IL-6/sIL-6R (50/250 ng/mL)

Figure 2 Effects of Dex on GAG loss and chondrocyte biosynthesis in bovine cartilage treated with combinations of mechanical injury,
TNFa and IL-6/sIL-6R. A) Percentage of GAG loss in bovine cartilage in response to six-day treatments. The mean ± SEM total GAG content was
466.3 ± 21.5 μg GAG/disk in the untreated control group. 10 nM DEX significantly reduced GAG loss from conditions involving TNFa plus IL-6/
sIL-6R, mechanical injury or both. B) Chondrocyte biosynthetic rates measured by
35
S-sulfate incorporation during days 4 to 6. TNFa, either with
or without IL-6/sIL-6R and mechanical injury, significantly lowered biosynthesis of proteoglycan, while the addition of DEX to these conditions
blocked the biosynthesis reductions. Values in A and B are presented as mean ± SEM. N = 10 cartilage disks in no-treatment control, DEX, TNFa,
and DEX + TNFa conditions. N = 5 cartilage disks in the remaining conditions.* = P < 0.05 (only comparisons from selected hypothesis are
shown). DEX, dexamethasone; GAG, glycosaminoglycans; IL-6, interleukin-6; SEM, standard error of the mean; sIL-6R, soluble interleukin-6 receptor;
TNFa, tumor necrosis factor alpha.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 7 of 15
rate; the addition of DEX significantly reduced the
release of GAGs and increased the sulfate incorporation
rate. The effects of DEX on biosynthesis were signifi-
cantly reversed by the pre sence of RU486, though the
increase in GAG release upon addition of RU486 was
not statistically significant. RU486 alone had no effect
on either normal controls or TNFa-treated samples.
Effects of DEX, TNFa and mechanical injury on
chondrocyte gene expression
Real-time qPCR was performed to determine bovine
chondrocyte gene expression responses t o four-day
treatments with DEX, TNFa and mechani cal injury
alone and in combinations (Figure 6). Matrix molec ules
collagen II and IX responded to both TNFa and TNFa
+ injury treatments with a significant decrease in mRNA
levels. DEX treatment increased the expression of both
genes in cartilage treated with TNFa to levels not signif-

icantly different than co ntrols. Aggrecan core protein
mRNA levels were significantly decreased in response to
TNFa + injury; however, the addition of DEX resulted
in mRNA levels not significantly different than controls.
IL-6 mRNA levels were increased significantly by treat-
ments involving TNFa, regardless of the presence of
DEX or mechanical injury treatment. T reatment condi-
tions had no significant effect on TNFa or IL-1b mRNA
levels.
Analysis also showed that TNFa alone significa ntly
up-regulated the levels of ADAMTS-4 and MMP-3
mRNA. TNFa + injury significantly increased
ADAMTS-4 and ADAMTS-5 mRNA levels in the pre-
sence of DEX. Additional genes, r elated to protease
and protease inhibition, were up-regulated in response
to the TNFa + injury trea tment, including TIMP-3
and MMP-3. Among the m atrix proteases, only MMP-
3 mRNA showe d reduced expression in response to
DEX + TNFa and DEX + TNFa+injurytreatments,
whereas ADAMTS-5 mRNA levels were not down-
regulated in the presence of DEX.
iNOS and nitrite
iNOS message expression was significantly elevated in
response to all treatments with TNFa , but not by injury
alone. The induction of iNOS mRNA was not abrogated
by the addition of DEX (Figure 7A). However, DEX sig-
nificantly reduced the amount of nitrite released to the
conditioned medium caused by TNFa treatment alone
(Figure 7B). Western blot analysis showed that iNOS
protein levels in the TNFa + DEX and TNFa + injury +

DEX conditions were markedly reduced compared to
these same conditions without DEX (Figure 7C).
Proprotein convertase (PC) inhibitor decreased GAG loss
induced by cytokine and mechanical injury treatments
To assess the role of PC in cartilage degradation, a gen-
eral PC inhibitor, decanoyl-RVKR-CMK, was added to
the conditions of TNFa,TNFa + IL-6/sIL-6R, and
TNFa + IL-6/sIL-6R + injury. 10 μM CMK significantly
decreased GAG release induced by TN Fa + IL-6/sIL-6R
+ injury (Figure 8).
0
10
20
30
40
50
GAG Loss from Human Knee
Cartilage (% of Total)
Dex (100nM)
+
++
*
T
NFα (100ng/mL)
IL-6/sIL-6R (50/250ng/mL)
Mechanical Injury
+
+
+
+

-
+
+
++
-
-
-
-
-
-
-
-
-
-
-
-
Figure 3 Effect of DEX on human knee cartilage treated with TNFa and TNFa in combination with injury and IL-6/sIL-6R .The
percentage of GAG loss was measured from 10-day treatments. All cartilage disks included superficial surface. The total GAG content was 168.9
± 17.1 μg GAG/disk in the untreated control group. 100 nM DEX significantly reduced GAG release induced by treatments with TNFa, IL-6/sIL-6R
and mechanical injury. In each condition, n = 6 cartilage disks. * = P < 0.05 (only comparisons from selected hypothesis are shown). DEX,
dexamethasone; GAG, glycosaminoglycans; IL-6, interleukin-6; sIL-6R, soluble interleukin-6 receptor; TNFa, tumor necrosis factor alpha.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 8 of 15
0
10
20
30
40
0 2 4
2-day DEX pre-treatment

No pre-treatment
TNFα-Induced GAG Loss
(% of Total)
Days
*
0
10
20
30
40
50
TNFα
No Pre-treatment
TNFα with
DEX Pre-treatmen
t
*
Day -2 to Day 0 Day 0 to Day 4
TNFα; No DEX
+/- DEX Pre-treatment
0
10
20
30
0 2 4
GAG Loss (% of Total)
Da
y
s
TNFα

TNFα+DEX
*
0
20
40
60
80
100
120
140
Sulfate Incorporation from
Days 2-4 (pmol/hr.mg)

Sulfate Incorporation from
Days 2-4 (pmol/hr.mg)

TNFα TNFα+DEX
Day -2 to Day 0
Day 0 to Day 4
TNFα +/- DEX Post-Treatment
TNFα
*
A. B.
C. D.
Figure 4 DEX Pre and post treatment. A, Cumulative GAG loss and B, sulfate incorporation (measured in the last two days) from bovine
cartilage samples pre-treated with 10 nM DEX for two days prior to a four-day TNFa treatment. On Day 4, Cartilage samples pre-incubated with
DEX released significantly less GAG, and showed significantly higher proteoglycan synthesis in the TNFa treatment compared to samples
without DEX pre-treatment. C, Cumulative GAG loss and D, sulfate incorporation (measured in the last two days) in bovine cartilage samples
treated with TNFa, in the presence or absence of 10 nM DEX, with a two-day pre-exposure to TNFa. DEX treatment introduced after the TNFa
pre-treatment showed significantly reduced GAG loss and increased sulfate incorporation on Day 4. In each condition, n = 5 cartilage disks. * = P

< 0.05 (only comparing the GAG loss difference between conditions on Day 4). DEX: dexamethasone; GAG: glycosaminoglycans; TNFa: tumour
necrosis factor alpha.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 9 of 15
Discussion
The objecti ve of this study was to determine the effe cts
of DEX on cartilage proteoglycan degradation and
synthesis in response to combined treatments with
mechanical injury and pro-inflammatory cytokines. We
previously reported that co-stimulatio n of cartilage with
TNFa and IL-6/sIL-6R caused significantly more GAG
release than either cytokine alone, in both immature
bovine knee and adult human knee and ankle cartilage
[11]. Moreover, mechanical injury substantially poten-
tiated the combined catabolic effects of TNFa and IL-6/
sIL-6R by inducing severe matrix degradation. In this
study, we first demonstrated that DEX, over a wide
range of concentrations (1 nM to 100 μM), completely
blocked TNFa-induced GAG loss and reversed the
reductioninbiosynthesiscausedbyTNFa in bovine
car tilage (Figure 1). Even in t he absence of cytokine sti-
mulation, cartilage disks exposed to higher concentra-
tions (that is, 0.1 to 100 μM) of DEX released fewer
GAGs and showed increased sulfate incorporation com-
pared to control samples.
Importantly, DEX (10 nM) also restored proteoglycan
biosynthesis and inhibited GAG loss caused by the treat-
ments with TNFa + IL-6/sIL-6R, injury + TNFa,and
injury + TNFa + IL-6/sIL-6R (Figures 2 and 3). The
proteoglycan fragments produced under these condi-

tions were previously found to be generated by aggreca-
nases, not MMPs [11]. Thus, the inhibitory effect of
0
5
10
15
20
T
NFα(25ng/mL)
−
−
+
+
+
+
GAG Loss
(% of Total)
*
0
30
60
90
120
150
Sulfate Incorporation
(pmol/hr.mg)
*
*
A.
B.

DEX(10nM)
RU (1μM)
−
−
−
−
−
+
−
−
+
+
+
+
TNFα(25ng/mL)
−
−
+
+
+
+
DEX(10nM)
RU (1μM)
−
−
−
−
−
+
−

−
+
+
+
+
*
*
Figure 5 The percentage of GAG loss in six days (A) and proteoglycan biosynthesis measured from days 4 to 6 in bovine cartilage in
response to TNFa, DEX and glucocorticoid receptor antagonist, RU 486 (B). RU reversed the effect of DEX in sulfate incorporation. In each
condition, n = 5 cartilage disks. * = P < 0.05 (only selected comparisons are shown). DEX, dexamethasone; GAG, glycosaminoglycans; RU486, a
glucocorticoid receptor antagonist; TNFa, tumor necrosis factor alpha.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 10 of 15
DEX on m atrix degradation may involve modulating the
proteolytic activities of aggrecanases. Recently, Malfait et
al. demonstrated that DEX blocked aggrecanase activity
in an in vivo model of cartilage degradation: intra-articu-
lar injection of TNFa in rats resulted in aggrecanase-
generated proteoglycan degradation, which could be
inhibited by either an aggrecanase inhibitor or DEX, but
not a non-steroidal anti-inflammatory drug [44].
Surprisi ngly, DEX did not abrogate GAG release via a
substantial reduction in aggrecanase transcriptional
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-

-
-
+
+
+
- ++
+
+
+
-
+
-
+
+
-
-
0
0.5
1
1.5
2
0
1
2
3
4
0
0.5
1
1.5

2
0
1
2
3
4
1
2
3
4
0.1
1
10
100
0
1
2
3
4
5
6
7
ADAMTS-4
0
2
4
6
8
10
12

0
1
2
3
4
5
6
Relative Expression
(Treatment/Control)
A
ggrecan
Relative Expression
(Treatment/Control)
Collagen II
Relative Expression
(Treatment/Control)
Relative Expression
(Treatment/Control)
Collagen IX
TNF-α
Relative Expression
(Treatment/Control)
Relative Expression
(Treatment/Control)
IL-1β
IL-6
Relative Expression
(Treatment/Control)
Relative Expression
(Treatment/Control)

ADAMTS-5
0.1
1
10
100
MMP-3
Relative Expression
(Treatment/Control)
Relative Expression
(Treatment/Control)
TIMP-3
***
***
***
*
**
*
*
*
*
*
*
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-

-
+
+
+
- +++
+
+
-
+
-
+
+
-
-
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-
-
+
+
+
-
++
+
+

+
-
+
-
+
+
-
-
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-
-
+
+
+
-
++
+
+
+
-
+
-
+
+

-
-
DEX (10nM)

TNFα (25ng/mL)
Mechanical Injury
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-
-
+
+
+
-
+
+
+
+
+
-
+
-
+
+
-

-
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-
-
+
+
+
- +++
+
+
-
+
-
+
+
-
-
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-

-
-
+
+
+
- +++
+
+
-
+
-
+
+
-
-
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-
-
+
+
+
- +++
+
+

-
+
-
+
+
-
-
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-
-
+
+
+
- +++
+
+
-
+
-
+
+
-
-
-

+
-
-
-
+
+
+
- ++
+
+
+
-
+
-
+
+
-
-
Figure 6 Changes in c hondrocyte gene expression after four-day treatments with DEX, TNFa, injury and their combinations. Values at
the y-axis represent the expression level of each gene normalized to that of the no-treatment control (y = 1, dotted line). Values are mean ±
SEM, n = 6 animals. * = P < 0.05 vs. no-treatment control. DEX: dexamethasone; SEM: standard error of the mean; TNFa: tumor necrosis factor
alpha.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 11 of 15
levels. In particular, the mRNA levels of ADAMTS-4
and -5 in response to TNFa + injury treatment
remained elevated in the presence of DEX (Figure 6).
Similarly, DEX did not down-regulate the gene expres-
sion of iNOS, although it markedly reduced the level of
iNOS protein as well as nitric oxide production in both

cytokine-stimulated and cytokine plus injury-treated car-
tilage (Figure 7). Previous studies by Guerne et al. [45]
and Shalom-Barak et al. [46] also reported the down-
regulation of cytokine-induced nitric oxide synthesis in
human chondrocytes by glucocorticoids. Therefore, DEX
may not regulate matrix degradation at the transcrip-
tional level alone. Aggrecanase activity can be affected
at multiple levels, including altered protein expression,
pro-enzyme activation and binding to aggrecan via the
C-terminal thrombospond in motif. In this study, we
hypothesized that DEX may block aggrecanase activity
by inhibiting the activation of latent pro-ADAMTS-4
0
5
10
15
20
25
30
35
40
Cumulative Nitrite Release (uM)
1
10
10 0
1000
10000
**
*
*

iNOS Gene Expression
(Treatment/Control)
A
.
B.
C.
*
DEX (10nM)

-
+
TNFα (25ng/mL)
Mechanical Injury
-
-
-
+
+
+
-
+
+
+
+
+
-
+
-
+
+

-
-
-
-
-
-
+
-
-
-
+
+
+
-
+
+
+
+
+
-
+
-
+
+
-
-
DEX (10nM)

TNFα (25ng/mL)
Mechanical Injury

+
+
+
+
+
-
+
-
+
+
-
-
DEX (10nM)

TNFα (25ng/mL)
Mechanical Injury
Figure 7 Effects of DEX on iNOS gene and protein expression and nitrite production. A. iNOS relative gene expression. Values at the y-axis
represent the expression level of each gene normalized to that of the no-treatment control (y = 1). Values are mean ± SEM, n = 6 animals. * = P
< 0.05 vs. no-treatment control. B. Nitrite released to the medium from days 0 to 4. In each condition, n = 6 cartilage disks. *= P < 0.05 (only
comparisons from selected hypotheses are shown). C. Western blot for iNOS protein. DEX, dexamethasone; iNOS, inducible nitric oxide synthase;
SEM, standard error of the mean.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 12 of 15
and -5. We showed that blocking PC activity signifi-
cantly reduced GAG loss in the cytokine plus injury
treatments (Figure 8), consistent with the important role
of PCs in proteoglycan degradation. Others have made
similar observations with TNFa-treated cartilage [26].
Ongoing studies focus on how DEX modulates PC activ-
ities as well as other possible mechanisms involved in

DEX-induced inhibition of proteoglycan degradation.
We further demonstrated that treating cartilage with
DEX either before or after TNFa stimulation significantly
reduced GAG loss and increased proteoglycan biosynth-
esis (Figure 4). These observations suggest that the effects
of DEX are long lasting and may provide protection
against further exposure to cytokines. Even when catabolic
processes have already begun in cartilage, DEX treatment
could still suppress GAG loss and increase biosynthesis.
In this study, we also observed that DEX (100 nM)
significantly reduced GAG loss in human cartilage
(though no stimulation of proteoglycan biosynthesis was
seen). Hardy et al. also observed that DEX blocked IL-1
stimulated proteoglycan degradation in OA cartilage
cultured with synovium [47]. Guerne et al. reported that
DEX inhibited the down-regulating effect of IL-1 and
IL-6/sIL-6R on proteoglycan synthesis, enhancing matrix
synthesis in normal, and, to a lesser extent in osteoar-
thritic human chondrocytes [45]. Together these reports
indicate DEX may also produce favorable responses in
human cartilage.
GCs have been widely used in the treatment of joint
diseases [12,13]. Most studies and trials reported benefi-
cial responses, including significantly greater reduction
of pain and tenderness, and increased motion in the
injected joi nt [48,49]. However, becau se the mechanism
of GCs in cartilag e function is not well understood, and
since there have been anecdotal reports of GC-related
side effects when treating joint diseases, the chronic use
of GCs in OA treatment remains controversial. It has

been noted that the reports describing negative effects
of GCs often involved either frequent injections or high
dosages [50]. More careful reviews have shown that the
efficacy of GC is dependent on the concentration used
[51]. In order to avoid complications, longer intervals
between GCs injections for the weight-bearing joints
have been recommended [52]. Future intra-articular
treatments may also involve the use of micro and nano
drug delivery technologies, which could enable local,
controlled release of GC and avoid the problems asso-
ciated with frequent injections and over dosage [53].
There have not been any reports on the long-term
effects of GC treatment on joint injury. However, the
current study suggests the concept that immediate treat-
ment of DEX in the injured knee may greatly retard the
initial progression of cartilage degradation. Moreover,
our data suggest that even delayed administration of
DEX may also be beneficial, thereby providing the clini-
cian with a window of therapeutic opportunity.
0
20
40
60
80
100
GAG Loss (% of Total)
CMK(10μM)
−− − −+++
*
TNFα (25ng/mL)

Mechanical Injury
IL-6/sIL-6R (50/250 ng/mL)
−
−−
−
−
+
+
+
+
+
+
+
+
+
+
−
+
+
−
−
−
Figure 8 Effects of CMK on GAG loss in treatment conditions. Percentage of GAG loss from bovine cartilage in response to treatments with
TNFa, IL-6/sIL-6R, mechanical injury and CMK, a general inhibitor for PCs. CMK significantly reduced GAG loss caused by TNFa + IL-6 +
mechanical injury. In each condition, n = 5 cartilage disks. *P < 0.05 (only comparisons from selected hypotheses are shown). CMK, decanoyl-
RVKR-CMK, a general proprotein convertase inhibitor; GAG, glycosaminoglycans; IL-6, interleukin 6; PC, proprotein convertase; sIL-6R, soluble
interleukin-6 receptor; TNFa, tumor necrosis factor alpha.
Lu et al. Arthritis Research & Therapy 2011, 13:R142
/>Page 13 of 15
Conclusions

Acute knee injury initiates cascades of catabolic events
in joint tissues, including mechanical disruption of carti-
lage matrix and increasing synovial fluid concentrations
of pro-inflammatory cytokines. Glucocorticoid treatment
of cartilage can effectively abolish matrix degradation
induced by the combination of pro-inflammatory cyto-
kines and injury. We suggest that DEX can protect car-
tilage matrix from post-traumatic degenerative changes
by both suppressing catabolic activities and maintaining
matrix biosynthesis. DEX inhibits catabolism of proteo-
glycans by modulating aggrecanase proteolytic activities
within cartilage. DEX, therefore, may be a promising
therapeutic agent for preventing cartilage degener ation
and post-traumatic osteoarthritis in individuals following
joint injury.
Abbreviations
ACL: anterior cruciate ligament; ADAMTS: a disintegrin and metalloproteinase
with thrombospondin motifs; CMK: decanoyl-RVKR-CMK, a general
proprotein convertase inhibitor; DEX: dexamethasone; DMEM: Dulbecco’s
Modified Eagle’s Medium; DMMB: dimethylmethylene blue; GAG:
glycosaminoglycans; GC: glucocorticoid; GR: glucocorticoid receptor; HEPES:
4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid; IL-6: interleukin-6; iNOS:
inducible nitric oxide synthase; MMP-3: matrix metalloproteinase-3; OA:
osteoarthritis; PACE: furin/paired basic amino-acid cleaving enzyme; PC:
proprotein convertase; qPCR: quantitative real time polymerase chain
reaction; RA: rheumatoid arthritis; rhTNFα: recombinant human tumor
necrosis factor alpha; RU486: a glucocorticoid receptor antagonist; SEM:
standard error of the mean; sGAG: sulfated glycosaminoglycans; sIL-6R:
soluble interleukin-6 receptor; TIMP-3: tissue inhibitor of metalloproteinase-3;
TNFα: tumor necrosis factor alpha.

Acknowledgements
Research supported by NIH/NIAMS grants AR045779 and AR033236 (AJG).
The authors thank Drs. Anna Plaas, Ada Cole and John Sandy of Rush
University, Chicago, for continued collaboration in enabling and
coordinating the use of human tissue.
Author details
1
Department of Biological Engineering, MIT, 500 Technology Square NE47-
377, Cambridge, MA, 02139, USA.
2
Department of Orthopaedic Surgery, Beth
Israel Deaconess Medical Center, 330 Brookline Avenue, RN-115, Boston, MA,
02215 USA.
3
Departments of Electrical Engineering and Computer Science
and Mechanical Engineering, MIT, 500 Technology Square NE47- 377,
Cambridge, MA, 02139, USA.
Authors’ contributions
YCL conducted all the experiments, analyzed the data, conceived of and
designed the studies, confirmed data analysis and wrote the manuscript. CE
and AJG conceived of and designed the studies, confirmed data analysis
and wrote the manuscript. All authors read and approve the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 22 March 2011 Revised: 6 May 2011
Accepted: 2 September 2011 Publi shed: 2 September 2011
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doi:10.1186/ar3456

Cite this article as: Lu et al.: Effects of short-term glucocorticoid
treatment on changes in cartilage matrix degradation and chondrocyte
gene expression induced by mechanical injury and inflammatory
cytokines. Arthritis Research & Therapy 2011 13:R142.
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